I. Live Bacterial Vector Vaccines
The advent of recombinant DNA technology has greatly accelerated the development of vaccines to control epidemic, endemic, and pandemic infectious diseases (Woodrow et al, New Generation Vaccines: The Molecular Approach, Eds., Marcel Dekker, Inc., New York, N.Y. (1989); Cryz, Vaccines and Immunotherapy, Ed., Pergamon Press, New York, N.Y. (1991); and Levine et al, Ped. Ann., 22:719-725 (1993)). In particular, this technology has enabled the growth of a new class of vaccines called bacterial vector vaccines (Curtiss, In: New Generation Vaccines: The Molecular Approach, Ed., Marcel Dekker, Inc., New York, N.Y., pages 161-188 and 269-288 (1989); and Mims et al, In: Medical Microbiology, Eds., Mosby-Year Book Europe Ltd., London (1993)). These vaccines can enter the host, either orally, intranasally or parenterally. Once gaining access to the host, the bacterial vector vaccines express an engineered prokaryotic expression cassette contained therein that encodes a foreign antigen(s). Foreign antigens can be any protein (or part of a protein) or combination thereof from a bacterial, viral, or parasitic pathogen that has vaccine properties (New Generation Vaccines: The Molecular Approach, supra; Vaccines and Immunotherapy, supra; Hilleman, Dev. Biol. Stand., 82:3-20 (1994);.degree. Formal et al, Infect. Immun. 34:746-751 (1981); Gonzalez et al, J. Infect. Dis., 169:927-931 (1994); Stevenson et al, FEMS Lett., 28:317-320 (1985); Aggarwal et al, J. Exp. Med., 172:1083-1090 (1990); Hone et al, Microbial. Path., 5:407-418 (1988); Flynn et al, Mol. Microbiol., 4:2111-2118 (1990); Walker et al, Infect. Immun., 60:4260-4268 (1992); Cardenas et al, Vacc., 11:126-135 (1993); Curtiss et al, Dev. Biol. Stand., 82:23-33 (1994); Simonet et al, Infect. Immun., 62:863-867 (1994); Charbit et al, Vacc., 11:1221-1228 (1993); Turner et al, Infect. Immun., 61:5374-5380 (1993); Schodel et al, Infect. Immun., 62:1669-1676 (1994); Schodel et al, J. Immunol., 145:4317-4321 (1990); Stabel et al, Infect. Immun., 59:2941-2947 (1991); Brown, J. Infect. Dis., 155:86-92 (1987); Doggett et al, Infect. Immun., 61:1859-1866 (1993); Brett et al, Immunol., 80:306-312 (1993); Yang et al, J. Immunol., 145:2281-2285 (1990); Gao et al, Infect. Immun., 60:3780-3789 (1992); and Chatfield et al, Bio/Technology, 10:888-892 (1992)). Delivery of the foreign antigen to the host tissue using bacterial vector vaccines results in host immune responses against the foreign antigen, which provide protection against the pathogen from which the foreign antigen originates (Mims, The Pathogenesis of Infectious Disease, Academic Press, London (1987); and New Generation Vaccines: The Molecular Approach, supra).
Of the bacterial vector vaccines, live oral Salmonella vector vaccines have been studied most extensively. There are numerous examples showing that Salmonella vectors are capable of eliciting humoral and cellular immunity against bacterial, viral and parasitic antigens (Formal et al, Infect. Immun., 34:746-751 (1981); Gonzalez et al, supra; Stevenson et al, supra; Aggarwal et al, supra; Hone et al, supra; Flynn et al, supra; Walker et al, supra; Cardenas et al, supra; Curtiss et al, supra; Simonet et al, supra; Charbit et al, supra; Turner et al, supra; Schodel et al, supra, Schodel et al (1990), supra; Stabel et al, supra; Brown, supra; Doggett et al, supra; Brett et al, supra; Yang et al, supra; Gao et al, supra; and Chatfield et al, supra). These humoral responses occur in the mucosal (Stevenson et al, supra; Cardenas et al, supra; Walker et al, supra; and Simonet et al, supra), and systemic compartments (Gonzalez et al, supra; Stevenson et al, supra; Aggarwal et al, supra; Hone et al, supra; Flynn et al, supra; Walker et al, supra; Cardenas et al, supra; Curtiss et al, supra; Simonet et al, supra; Charbit et al, supra; Turner et al, supra; Schodel et al, supra, Schodel et al (1990), supra; Stabel et al, supra; Brown, supra; Doggett et al, supra; Brett et al, supra; Yang et al, supra; Gao et al, supra; and Chatfield et al, supra). Live oral Salmonella vector vaccines also elicit T cell responses against foreign antigens (Wick et al, Infect. Immun., 62:4542-4548 (1994)). These include antigen-specific cytotoxic CD8.sup.+ T cell responses (Gonzalez et al, supra; Aggarwal et al, supra; Flynn et al, supra; Turner et al, supra; and Gao et al, supra).
Ideally, bacterial vector vaccines are genetically defined, attenuated and well-tolerated by the recipient animal or human, and retain immunogenicity (Hone et al, Vaccine, 9:810-816 (1991); Tacket et al, Infect. Immun., 60:536-541 (1992); Hone et al, J. Clin. Invest., 90:412-420 (1992); Chatfield et al, Vaccine, 10:8-11 (1992); Tacket et al, Vaccine, 10:443-446 (1992); and Mims, supra). Recently, the number of potential bacterial vector vaccines for the delivery of prokaryotic expression cassettes has grown. They now include, but are not restricted to Yersinia enterocolitica (van Damme et al, Gastroenterol., 103:520-531 (1992)), Shigella spp. (Noriega et al, Infect. Immun., 62:5168-5172 (1994)), Vibrio cholerae (Levine et al, In: Vibrio cholerae, Molecular to Global Perspectives, Wachsmuth et al, Eds, ASM Press, Washington, D.C., pages 395-414 (1994)), Mycobacterium strain BCG (Lagranderie et al, Vaccine, 11:1283-1290 (1993); Flynn, Cell. Molec. Biol., 40(Suppl. 1):31-36 (1994)), and Listeria monocytogenes (Schafer et al, J. Immunol., 149:53-59 (1992)) vector vaccines.
II. Eukaryotic and Prokaryotic Expression Cassettes
Normally, an expression cassette is composed of a promoter region, a transcriptional initiation site, a ribosome binding site (RBS), an open reading frame (orf) encoding a protein (or fragment thereof), with or without sites for RNA splicing (only in eukaryotes), a translational stop codon, a transcriptional terminator and post-transcriptional poly-adenosine processing sites (only in eukaryotes) (Wormington, Curr. Opin. Cell Biol., 5:950-954 (1993); Reznikoff et al, Maximizing Gene Expression, Eds., Butterworths, Stoneham, Mass. (1986)). The promoter region, the RBS, the splicing sites, the transcriptional terminator and post-transcriptional poly-adenosine processing sites are markedly different in eukaryotic expression cassettes than those found in prokaryotic expression cassettes (Wasylyk, In: Maximizing Gene Expression, supra, pages 79-99; Reznikoff et al, In: Maximizing Gene Expression, supra, pages 1-34; and Lewin, Genes V, Oxford University Press, Oxford (1994)). These differences prevent expression of prokaryotic expression cassettes in eukaryotic cells and visa versa (Miller et al, Mol. Micro., 4:881-893 (1990); and Williamson et al, Appl. Env. Micro., 60:771-776 (1994)).
Prokaryotic promoters are not active in eukaryotic cells and eukaryotic promoters are not active in prokaryotic cells (Eick et al, Trends in Genetics, 10:292-296 (1994)). The basis for the functional diversity of eukaryotic versus prokaryotic promoters is mediated by RNA-polymerase, transcription regulatory factors and the DNA structure of the promoter (Eick et al, supra).
RNA polymerases are DNA-binding proteins that recognize specific sequences in the DNA of promoter regions. RNA polymerases catalyze the synthesis of RNA molecules by polymerizing nucleoside triphosphates in the specific order that is dictated by the DNA coding sequence (Libby et al, Mol. Micro., 5:999-1004 (1991); Kerppola et al, FASEB J., 5:2833-2842 (1991); Alberts et al, Molecular Biology of the Cell, Eds., Garland Publishing Inc, New York, N.Y. (1994); Watson et al, Molecular Biology of the Gene, Vol. 1, Eds., The Benjamin/Cummings Publishing Comp. Inc., Menlo Park Calif. (1987); and Lewin, supra). RNA polymerases of prokaryotes typically are composed of two identical .alpha. subunits and two similar, but non-identical, .beta. and .beta.' subunits (Ishihama, Mol. Micro., 6:3283-3288 (1992); Watson et al, supra; Alberts et al, supra; and Lewin, supra). The specificity of prokaryotic RNA polymerases for a given promoter region is mediated by specific a factors that recognize core sequences encoded by the DNA in the promoter regions (Libby et al, supra; and Lewin, supra).
In eukaryotic cells, there are three RNA polymerases. Each of the different RNA polymerases contain 10 to 15 different subunits and each performs a different function (Kerppola et al, supra; Larson et al, Biochem. Cell. Biol., 69:5-22 (1991); Archambault et al, Microbiol. Rev., 57:703-724 (1993); Watson et al, supra; Alberts et al, supra; and Lewin, supra). In addition, specific DNA-binding factors may be required for the association of eukaryotic RNA polymerases to the DNA in a promoter region (Darnell et al, Molecular Cell Biology, Scientific American Books, Inc., W. H. Freeman and Co., New York, N.Y. (1990); Hori et al, Curr. Opin. Gen. Devel., 4:236-244 (1994); Lewin, supra; Watson et al, supra; and Alberts et al, supra). These binding factors recognize specific sequences in the DNA, and also interact with the eukaryotic RNA polymerases.
There is little similarity between the primary DNA sequence of prokaryotic promoters and the primary DNA sequence of eukaryotic promoters. The DNA structure of prokaryotic promoters is relatively simple, consisting of -10 and -35 regions and proximal regulatory sequences (Darnell et al, supra; Lewin, supra; Watson et al, supra; and Alberts et al, supra) Prokaryotic promoters are located upstream of the transcription start site. Eukaryotic promoters, in contrast, are composed of a core unit, which is usually located from 50 bp upstream to 20 bp downstream of the transcriptional start site (Darnell et al, supra; Lewin, supra; Watson et al, supra; and Alberts et al, supra), and enhancer sequences that are located from about 100 to 200 bp upstream of the transcriptional start, but also can be located in distal locations (Sonenberg, Curr. Opin. Gen. Devel., 4:310-315 (1994); Geballe et al, Trends Bio. Sci., 1:159-164 (1994); and Lewin, supra).
The RBS is the site recognized by the ribosome for binding to the 5' end of messenger RNA (mRNA) molecules. This binding is essential for the translation of mRNA into a protein by the ribosome. In prokaryotes, the RBS in the 5' end of the mRNA molecule is a sequence that is complementary to the 3' end of the small ribosomal RNA molecule (5S rRNA) (Chatterji et al, Ind. J. Biochem. Biophys., 29:128-134 (1992); and Darnell et al, supra; Lewin, supra; Watson et al, supra; and Watson et al, supra). The presence of the RBS promotes binding of the ribosome to the 5' end of the nascent mRNA molecule. Translation is then initiated at the first AUG codon encountered by the scanning ribosome (Darnell et al, supra; Lewin, supra; Watson et al, supra; and Alberts et al, supra). At present, no such recognition pattern has been observed in eukaryotic mRNA-ribosome interactions (Eick et al, supra). In addition, prior to initiation of translation of eukaryotic mRNA, the 5' end of the mRNA molecule is "capped" by addition of methylated guanylate to the first mRNA nucleotide residue (Darnell et al, supra; Lewin, supra; Watson et al, supra; and Alberts et al, supra). It has been proposed that recognition of the translational start site in mRNA by the eukaryotic ribosomes involves recognition of the cap, followed by binding to specific sequences surrounding the initiation codon on the mRNA. It is possible for cap independent translation initiation to occur and/or to place multiple eukaryotic coding sequences within a eukaryotic expression cassette if a cap-independent translation enhancer (CITE) sequence, such as derived from encephalomyocarditis virus (Duke et al, J. Virol., 66:1602-1609 (1992)), is included prior to or between the coding regions. However, the initiating AUG codon is not necessarily the first AUG codon encountered by the ribosome (Louis et al, Molec. Biol. Rep., 13:103-115 (1988); and Voorma et al, Molec. Biol. Rep., 19:139-145 (1994); Lewin, supra; Watson et al, supra; and Alberts et al, supra). Thus, RBS binding sequences in eukaryotes are sufficiently divergent from that of prokaryotic RBS such that the two are not interchangeable.
Termination of transcription in prokaryotes is mediated by rho-independent and rho-dependent stem loops (Richardson, Crit. Rev. Biochem. Mol. Biol., 28:1-30 (1993); Platt, Molec. Microbiol., 11:983-990 (1994); and Lewin, supra). In contrast, terminator loops are not commonly found downstream of eukaryotic expression cassettes, and transcription often extends beyond the end of the open reading frame (Tuite et al, Mol. Biol. Reps., 19:171-181 (1994)). However, usually the over-extended mRNA is specifically cleaved by endonucleases, and the 3' end of the mRNA is poly-adenylated by poly-A polymerase (Proudfoot, Cell, 64:671-674 (1991); Wahle, Bioassays, 14:113-118 (1992); Lewin, supra; and Watson et al, supra). Sequences that are recognized by the endonucleases and poly-A polymerase must be included in the 3' end of the mRNA molecule for these post-translation modifications to occur. Polyadenylation of the 3' end of mRNA molecules is thought to be a necessary step prior to transport of mRNA to the cytoplasm of eukaryotic cells and translation into proteins (Sachs et al, J. Biol. Chem., 268:22955-22958 (1993); and Sachs, Cell, 74:413-421 (1993)). A eukaryotic expression cassette does not need to code for a functional gene product, i.e., it may also code for a partial gene product which acts as an inhibitor of a eukaryotic enzyme (Warne et al, Nature, 364:352-355 (1993); and Wang, J. Cell Biochem., 45:49-53 (1991)), an antisense RNA (Magrath, Ann. Oncol., 5(Suppl 1):67-70 (1994); Milligan et al, Ann. NY Acad. Sci., 716:228-241 (1994); and Schreier, Pharma. Acta Helv., 68:145-159 (1994)), a catalytic RNA (Cech, Biochem. Soc. Trans., 21:229-234 (1993); Cech, Gene, 135:33-36 (1993); Long et al, FASE J., 7:25-30; and Rosi et al, Pharm. Therap., 50:245-254 (1991)), or any other sequence which can be transcribed into RNA.
Due to a need for eukaryotic expression cassettes to study the biology of eukaryotic cells, a number of eukaryotic expression plasmids are now readily available. These include, but are not limited to, commercial products from companies, such as Invitrogen Corporation (San Diego, Calif.), Stratagene (La Jolla, Calif.), ClonTech (Palo Alto, Calif.) and Promega Corporation (Madison, Wis.). All of these plasmids contain the elements of a eukaryotic expression cassette listed above, and many also contain a prokaryotic promoter sequence such that the gene placed downstream from the promoter sequences will be expressed in a prokaryotic as well as in an eukaryotic cell, e.g., plasmid pSV.beta.-gal contains the eukaryotic SV40 promoter and the prokaryotic gpt promoter which allows for the expression of the .beta.-galactosidase gene in either prokaryotic cells or eukaryotic cells (Promega Corp.).
III. Delivery of Eukaryotic Expression Cassettes to Plant Cells
The use of Agrobacterium tumerfacium Ti plasmid to stably deliver DNA to plant cells has been a fundamental component of the boom in plant biotechnology. This system is unique in that it uses a pilus-like structure which binds to the plant cell via specific receptors, and then through a process that resembles bacterial conjugation, delivers the Ti plasmid-linked DNA to the plant cell (Zambryski, Ann. Rev. Genet., 22:1-30 (1988); Lessl et al, Cell, 77:321-324 (1994); and Walden et al, Eur. J. Biochem., 192:563-576 (1990)). The specificity of this delivery system for plants, however, renders it ineffective for delivery of DNA to animal cells (Chilton, Proc. Natl. Acad. Sci., USA, 90:3119-3120 (1993); and Walden et al, supra).
IV. Delivery of Eukaryotic Expression Cassettes to Animal Cells
There are several techniques for introducing DNA into animal cells cultured in vitro. These include chemical methods (Felgner et al, Proc. Natl. Acad. Sci., USA, 84:7413-7417 (1987); Bothwell et al, Methods for Cloning and Analysis of Eukaryotic Genes, Eds., Jones and Bartlett Publishers Inc., Boston, Mass. (1990), Ausubel et al, Short Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y. (1992); and Farhood, Annal. N.Y. Acad. Sci., 716:23-34 (1994)), use of protoplasts (Bothwell, supra) or electrical pulses (Vatteroni et al, Mutn. Res., 291:163-169 (1993); Sabelnikov, Prog. Biophys. Mol. Biol., 62:119-152 (1994); Brothwell et al, supra; and Ausubel et al, supra), use of attenuated viruses (Moss, Dev. Biol. Stan., 82:55-63 (1994); and Brothwell et al, supra), as well as physical methods (Fynan et al, supra; Johnston et al, Meth. Cell Biol, 43(Pt A):353-365 (1994); Brothwell et al, supra; and Ausubel et al, supra).
Successful delivery of DNA to animal tissue has been achieved by cationic liposomes (Watanabe et al, Mol. Reprod. Dev., 38:268-274 (1994)), direct injection of naked DNA into animal muscle tissue (Robinson et al, Vacc., 11:957-960 (1993); Hoffman et al, Vacc., 12:1529-1533; (1994); Xiang et al, Virol., 199:132-140 (1994); Webster et al, Vacc., 12:1495-1498 (1994); Davis et al, Vacc., 12:1503-1509 (1994); and Davis et al, Hum. Molec. Gen., 2:1847-1851 (1993)), and embryos (Naito et al, Mol. Reprod. Dev., 39:153-161 (1994); and Burdon et al, Mol. Reprod. Dev., 33:436-442 (1992)), or intradermal injection of DNA using "gene gun" technology (Johnston et al, supra). A limitation of these techniques is that they only efficiently deliver DNA to parenteral sites. At present, effective delivery of eukaryotic expression cassettes to mucosal tissue has been met with limited success. This is presumably due to poor access to these sites, toxicity of the delivery vehicles or instability of the delivery vehicles when delivered orally.
The commercial application of DNA delivery technology to animal cells is broad and includes delivery of vaccine antigens (Fynan et al, Proc. Natl. Acad. Sci., USA, 90:11478-11482 (1993)), immunotherapeutic agents, and gene therapeutic agents (Darris et al, Cancer, 74(3 Suppl.):1021-1025 (1994); Magrath, Ann. Oncol., 5(Suppl 1):67-70 (1994); Milligan et al, Ann. NY Acad. Sci., 716:228-241 (1994); Schreier, Pharma. Acta Helv., 68:145-159 (1994); Cech, Biochem. Soc. Trans., 21:229-234 (1993); Cech, Gene, 135:33-36 (1993); Long et al, FASEB J., 7:25-30 (1993); and Rosi et al, Pharm. Therap., 50:245-254 1991)).
The delivery of endogenous and foreign genes to animal tissue for gene therapy has shown significant promise in experimental animals and volunteers (Nabel, Circulation, 91:541-548 (1995); Coovert et al, Curr. Opin. Neuro., 7:463-470 (1994); Foa, Bill. Clin. Haemat., 7:421-434 (1994); Bowers et al, J. Am. Diet. Assoc., 95:53-59 (1995); Perales et al, Eur. J. Biochem., 226:255-266 (1994); Danko et al, Vacc., 12:1499-1502 (1994); Conry et al, Canc. Res., 54:1164-1168 (1994); and Smith, J. Hemat., 1:155-166 (1992)). Recently, naked DNA vaccines carrying eukaryotic expression cassettes have been used to successfully immunize against influenza both in chickens (Robinson et al, supra) and ferrets (Webster et al, Vacc., 12:1495-1498 (1994)); against Plasmodium yoelii in mice (Hoffman et al, supra); against rabies in mice (Xiang et al, supra); against human carcinoembryonic antigen in mice (Conry et al, supra) and against hepatitis B in mice (Davis et al, supra). These observations open the additional possibility that delivery of endogenous and foreign genes to animal tissue could be used for prophylactic and therapeutic applications.
Therefore, there is a need to deliver eukaryotic expression cassettes, encoding endogenous or foreign genes that are vaccines or therapeutic agents to animal cells or tissue. In particular, a method that delivers eukaryotic expression cassettes to mucosal surfaces is highly desirable. Bacterial vector vaccines have been used in the past to deliver foreign antigens encoded on prokaryotic expression cassettes to animal tissue at mucosal sites.
The present invention describes a novel and unexpected finding that invasive bacteria are capable of delivering eukaryotic expression cassettes to animal cells and tissue. An important aspect of using live invasive bacteria to deliver eukaryotic expression cassettes is that they are capable of delivering DNA to mucosal sites.
Heretofore, there has been no documented demonstration of live bacteria invading animal cells and introducing a eukaryotic expression cassette(s), which then is expressed by the infected cells and progeny thereof. That is, the present invention provides the first documentation of genetic exchange between live invasive bacteria and animals cells. Heretofore, foreign antigen delivery by live bacterial vector vaccines merely involved delivery of prokaryotic expression cassettes to and expression of the foreign antigen by the bacterial vaccine vector, in animal cells or tissues. In contrast, the present invention involves the delivery of eukaryotic expression cassettes by live bacterial strains to animal cells in vitro or to cells in animal tissue, and expression of the eukaryotic expression cassettes by the animal cell or cells in animal tissue.